Immunofluorescent analysis of antibodies on kidney sections (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Andrew McMahon; Tracy Tran

Keywords

[‘antibodies’, ‘human renal cortex’, ‘nephrogenesis’, ‘kidney development’, ‘nephron progenitor’, ‘nephron’]

Subjects

[‘Cell biology’, ‘Immunological techniques’, ‘Gene expression analysis’]

Release Date

2016-08-01

Abstract

TBD

Procedure

Preparation of Sections:

Day 1:

1. Mouse kidneys are dissected into cold PBS from E15.5 Swiss-Webster embryos or P2 Swiss-Webster pups. Week 8 and Week 16 human fetal kidneys are dissected out from donated tissues.
2. Mouse kidneys are fixed in cold 4% paraformaldehyde (PFA) in PBS for 15 minutes on ice. Week 8 human kidneys undergo fixation in cold 4% PFA for 45 minutes at 4C. The fixation time for week 16 human kidneys is overnight at 4C.
3. The tissues are washed twice in PBS (5 minutes each).
4. Samples are cryopreserved in 30% sucrose in PBS with rocking at 4oC overnight.

Day 2:

1. Kidneys are swirled in Optimal Cutting Temperature (OCT) compound 3 times before embedding in OCT block. The OCT blocks with tissues are frozen on a dry ice-ethanol slurry.
2. Blocks are stored at -80oC, or 12-μm sections can be obtained freshly using a cryostat. Slides are stored at -80oC.

Immunofluorescence:

Day 1:

1. Slides are thawed and let dry at room temperature (RT).
2. Wash off OCT from the sections by placing slides in PBS for 5 minutes.
3. The sections are blocked using 1.5% SEA BLOCK block buffer (Thermo Fisher Scientific 37527) for 1 hour at RT.
4. Dilute primary antibodies in block buffer and incubate slides with primary antibody solution at 4oC overnight.

Day 2:

1. Wash the slides three times in PBS + 0.25% Triton-X, 5 minutes each wash.
2. Incubate sections in appropriate secondary antibodies at desired dilution in block buffer for 1 hour at RT.
3. Wash three times with in PBS + 0.25% Triton-X, 5 minutes each wash.
4. Rinse the sections once with PBS.
5. Dilute Hoechst 33342 in PBS (1:10000) and incubate the slides in Hoechst solution.

Consortium

(Re)Building a Kidney (RBK) Consortium